Mycoplasmas are infective agents of humans, laboratory, domestic and farm animals, plants, insects and cell cultures. Approximately 15% of continuous cell lines contain mycoplasmas. Virtually every cell culture parameter is affected. Mycoplasmal infection invalidates results of in vitro diagnostic and experimental procedures and wastes millions of federal grant dollars annually. The problem will increase as new types of non-fibroblast cell cultures are being propagated in a variety of media, including serum-free formulations and in large scale operations. Data are critically needed to determine the nature of mycoplasmas in this new generation of cell culture systems. This application addresses certain of these areas in detail: lymphoblastoid cells, cell cultures grown in serum-free media and detection methods for large scale growth of cell cultures. Cell culture systems can also represent valuable models to study mycoplasmal physiology and pathogenesis. Mycoplasma, Ureaplasma and Spiroplasma species do not propagate in cell culture media. They do grow, however, in cell cultures, suggesting the cells produce myocplasma growth factors. These studies will characterize these factors as well as define mechanisms whereby M. hyorhinis can grow in a T lymphocyte cell line in serum-free media without supplemental cholesterol, recently observed in this laboratory. Cell and organ culture studies with ureaplasmas will be expanded. Studies on the transformation of NIH 3T3 cells by a helical mycoplasma, Spiroplasma mirum, with high efficiency will be expanded, hopefully to define cellular and molecular mechanisms. An immunobinding technique developed in this laboratory to identify cell culture isolates offers potential to detect mycoplasmas in clinical specimens. These studies will be expanded. Studies contained in this proposal will generate significant information that will have practical and immediate aplication to specialists in cell culture as well as infectious disease. Results should contribute to improved detection methods, elucidation of the role mycoplasmas play in disease, and to a better understanding of the mechanisms of mycoplasmal-mammalian cell relationships, especially those that occur in serum free condition in vivo and in vitro.